The Wnt, TGF-β, and Notch signaling pathways are essential for the regulation of cellular polarity, differentiation, proliferation, and migration. Differential activation and mutual crosstalk of these pathways during animal development are crucial instructive forces in the initiation of the body axis and the development of organs and tissues. Due to the ability to initiate cell proliferation, these pathways are vulnerable to somatic mutations selectively producing cells, which ultimately slip through cellular and organismal checkpoints and develop into cancer. The architecture of the Wnt, TGF-β, and Notch signaling pathways is simple. The transmembrane receptor, activated by the extracellular stimulus, induces nuclear translocation of the transcription factor, which subsequently changes the expression of target genes. Nevertheless, these pathways are regulated by a myriad of factors involved in various feedback mechanisms or crosstalk. The most prominent group of regulators is the ubiquitin-proteasome system (UPS). To open the door to UPS-based therapeutic manipulations, a thorough understanding of these regulations at a molecular level and rigorous confirmation in vivo are required. In this quest, mouse models are exceptional and, thanks to the progress in genetic engineering, also an accessible tool. Here, we reviewed the current understanding of how the UPS regulates the Wnt, TGF-β, and Notch pathways and we summarized the knowledge gained from related mouse models.

• Klíčová slova
• cancer, gene inactivation, mouse model, ubiquitin–proteasome system,
• MeSH
• beta-katenin metabolismus   MeSH
• buněčná diferenciace fyziologie   MeSH
• homeostáza genetika   MeSH
• ligasy metabolismus   MeSH
• myši embryologie   genetika   MeSH
• proliferace buněk fyziologie   MeSH
• proteiny Wnt metabolismus   MeSH
• receptory Notch metabolismus   MeSH
• signální dráha Wnt fyziologie   MeSH
• transformující růstový faktor beta metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• ubikvitinligasy metabolismus   fyziologie   MeSH
• vývojová regulace genové exprese genetika   MeSH
• zvířata MeSH
• Check Tag
• myši embryologie   genetika   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• beta-katenin MeSH
• ligasy MeSH
• proteiny Wnt MeSH
• receptory Notch MeSH
• transformující růstový faktor beta MeSH
• transkripční faktory MeSH
• ubikvitin MeSH
• ubikvitinligasy MeSH

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.

• Klíčová slova
• endosome, phosphoinositide, vesicular traffic,
• MeSH
• 1-fosfatidylinositol-4-kinasa antagonisté a inhibitory   chemie   metabolismus   MeSH
• biologický transport MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endozomy účinky léků   metabolismus   MeSH
• Golgiho aparát účinky léků   metabolismus   MeSH
• HEK293 buňky MeSH
• inhibitory enzymů metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• preklinické hodnocení léčiv MeSH
• rychlé screeningové testy * MeSH
• simulace molekulového dockingu MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 1-fosfatidylinositol-4-kinasa MeSH
• inhibitory enzymů MeSH

Specificity of membrane fusion in vesicular trafficking is dependent on proper subcellular distribution of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Although SNARE complexes are fairly promiscuous in vitro, substantial specificity is achieved in cells owing to the spatial segregation and shielding of SNARE motifs prior to association with cognate Q-SNAREs. In this study, we identified phosphatidylinositol 4-kinase IIα (PI4K2A) as a binding partner of vesicle-associated membrane protein 3 (VAMP3), a small R-SNARE involved in recycling and retrograde transport, and found that the two proteins co-reside on tubulo-vesicular endosomes. PI4K2A knockdown inhibited VAMP3 trafficking to perinuclear membranes and impaired the rate of VAMP3-mediated recycling of the transferrin receptor. Moreover, depletion of PI4K2A significantly decreased association of VAMP3 with its cognate Q-SNARE Vti1a. Although binding of VAMP3 to PI4K2A did not require kinase activity, acute depletion of phosphatidylinositol 4-phosphate (PtdIns4P) on endosomes significantly delayed VAMP3 trafficking. Modulation of SNARE function by phospholipids had previously been proposed based on in vitro studies, and our study provides mechanistic evidence in support of these claims by identifying PI4K2A and PtdIns4P as regulators of an R-SNARE in intact cells.

• Klíčová slova
• PI4K2A, PtdIns4P, SNARE, Sorting, VAMP3, Vesicle fusion,
• MeSH
• buněčná membrána metabolismus   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• endozomy metabolismus   MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem metabolismus   MeSH
• fúze membrán fyziologie   MeSH
• lidé MeSH
• membránový protein 3 asociovaný s vezikuly metabolismus   MeSH
• proteiny SNARE metabolismus   MeSH
• receptory transferinu metabolismus   MeSH
• transport proteinů fyziologie   MeSH
• vedlejší histokompatibilní antigeny MeSH
• vezikulární transportní proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• membránový protein 3 asociovaný s vezikuly MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• proteiny SNARE MeSH
• receptory transferinu MeSH
• vedlejší histokompatibilní antigeny MeSH
• vezikulární transportní proteiny MeSH

β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.

• Klíčová slova
• Amer1/WTX/FAM123B, Dvl, Lrp6 Phosphorylation, Membrane Lipids, Phosphatidylinositol Kinase, Phosphatidylinositol Phosphate Kinase, Phosphatidylinositol Signaling, Wnt Signaling, β-Arrestin, β-Catenin,
• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• arrestiny genetika   metabolismus   MeSH
• beta arrestiny MeSH
• buněčná membrána metabolismus   MeSH
• embryo savčí cytologie   MeSH
• fibroblasty cytologie   metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• LDL receptor related protein 6 genetika   metabolismus   MeSH
• lidé MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové supresorové proteiny genetika   metabolismus   MeSH
• protein dishevelled MeSH
• protein Wnt3A genetika   metabolismus   MeSH
• RNA interference MeSH
• vazba proteinů MeSH
• vedlejší histokompatibilní antigeny MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-phosphatidylinositol-4-phosphate 5-kinase MeSH Prohlížeč
• adaptorové proteiny signální transdukční MeSH
• AMER1 protein, human MeSH Prohlížeč
• arrestiny MeSH
• beta arrestiny MeSH
• fosfatidylinositol-4,5-difosfát MeSH
• fosfoproteiny MeSH
• fosfotransferasy s alkoholovou skupinou jako akceptorem MeSH
• LDL receptor related protein 6 MeSH
• LRP6 protein, human MeSH Prohlížeč
• nádorové supresorové proteiny MeSH
• phosphatidylinositol phosphate 4-kinase MeSH Prohlížeč
• protein dishevelled MeSH
• protein Wnt3A MeSH
• vedlejší histokompatibilní antigeny MeSH